Rapid thermal cycling for sample analyses and processing

ABSTRACT

An apparatus for thermal processing of nucleic acid in a thermal profile. The apparatus employs a reactor holder for holding reactors to accommodate reaction material containing nucleic acid. The apparatus includes at least two baths separated by thermally insulating partition plate(s) where bath mediums are each maintainable at a predetermined temperature; and a transfer means for allowing the reactors to change position once or plurality of times between any two adjacent baths by selectively opening the partition plate(s) and without lifting the reactors out of the baths.

CROSS REFERENCE TO THE RELATED APPLICATIONS

The present application is continuous in part application of theInternational Patent Application No: PCT/SG2017/050285 filed on 6 Jun.2017, which claims priority to U.S. Patent Application No. 62/348,155filed on 10 Jun. 2016 and SG Patent Application No. 10201700260X filedon 12 Jan. 2017, the entire contents of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method and an apparatus forperforming amplification reaction of nucleic acids in a sample.

BACKGROUND

Polymerase chain reaction (PCR) is increasingly important to molecularbiology, food safety and environmental monitoring. A large number ofbiological researchers use PCR in their work on nucleic acid analyses,due to its high sensitivity and specificity. The time cycle of a PCR istypically in the order of an hour, primarily due to a time-consuming PCRthermal cycling process that is adapted to heat and cool reactorscontaining the sample to different temperatures for DNA denaturation,annealing and extension. Typically, the thermal cycling apparatus andmethod employ moving the reactors between two heating baths whosetemperatures are set at the target temperatures as required for nucleicacid amplification reactions. Researchers have been constantly strivingto increase the speed of thermal cycling.

Thermoelectric cooler (TEC) or Peltier cooler is often used as theheating/cooling element. However, it provides a typical ramping rate of1-5 degree C./sec which is rather slow in changing the temperature ofthe reactor and disadvantageously increases the time of the thermalcycling.

As an attempt to increase the PCR speed by reducing thermal mass,microfabricated PCR reactor with embedded thin film heater and sensorwas developed to achieve faster thermal cycling at a cooling rate of 74degree Celsius/s and a heating rate of around 60-90 degree Celsius/s.However, such a wafer fabrication process for making the PCR device isextremely expensive and thus is impractical in meeting the requirementof low cost disposable applications in biological testing.

Hot and cold air alternately flushing the reactors in a closed chamberto achieve higher temperature ramping than the TEC-based thermal cyclerhas been described. However, from the heat transfer point of view, airhas much lower thermal conductivity and heat capacity than liquid, hencethe temperature ramping of the air cycler is slower than that with aliquid. The TEC needs a significant amount of time to heat and coolitself and the heat block above the TEC. Further there is also need toovercome the contact thermal resistance between the heat block and thereactors.

Alternating water flushing cyclers were also developed in which water oftwo different temperatures alternately flush the reactors to achieve PCRspeed. However, such devices contain many pumps, valves and tubingconnectors which increase the complexity of maintenance and lower thereliability while dealing with high temperature and high pressure. Withcirculating liquid bath medium, the liquid commonly spills out from thebaths.

Traditional water bath PCR cyclers utilize the high thermal conductivityand heat capacity of water to achieve efficient temperature heating andcooling. But, such cyclers have large heating baths containing a largevolume of water which is hard to manage in loading and disposal, andalso makes the heating time to target temperatures too long beforethermal cycling can start. Such cyclers also have large device weightand high power consumption. The water tends to vaporize with usage andneeds to be topped up. Besides, during the thermal cycling every timethe reactor is alternately inserted into the baths, a layer of waterremains adhered on the reactor body when taken out of each bath, therebycausing the change in temperature inside the reactor to get slowerundesirably.

Researchers also tested moving heated rollers of different temperaturesto alternately contact the reactors. However, use of long tubingreactors make it not only cumbersome to install and operate a largearray of reactors, but also expensive. When the reactors are in a largearray or a panel, it may be challenging to achieve heating uniformityamong all the reactors.

FIG. 1A shows a schematic view of a portion of a typical thermal cyclingapparatus for thermal cycling of nucleic acid such as for PCR, primerextension or other enzymatic reactions. The apparatus has two baths 50and 51 each containing the bath medium 75, a bath heater 17 and a bathtemperature sensor 39 mounted along the bath surface to enable controlof the temperature of the bath medium 75. The bath 50 is suitable forthe step of denaturation and the bath 51 is suitable for the step ofannealing and/or extension. The bath medium 75 is liquid. For someembodiments, the bath heater 17 on the low temperature bath 51 isoptional, if bath 51 does not have to be heated. For the thermalcycling, the reactor 15 is alternately transferred between the baths 50,51 multiple times. The reactor 15 is sealed with a sealant or a cap 77and a portion of the reactor 15 herein is transparent to allow light topass through for dye or probe excitation and fluorescence imaging. Thearrangement for the fluorescent imaging may be in any form as in theart. Herein, the bath 51 has a transparent window 25 so thatillumination from the illumination source 44 reaches the reactor 15inside and the emitted beam is received by the receiver 43. Atemperature monitoring unit 34 is installed on the reactor holder 33 andmoves along with the reactor 15 between the baths 50, 51. Thetemperature monitoring unit 34 may be a fast response temperature sensor38 inserted into water or oil or a layer of oil over water 22 andsealed. Although only one reactor 15 is shown, according to otherembodiments the reactor holder 33 may accommodate a plurality ofreactors 15. The reactor transfer mechanism 85 transfers the reactor 15and the temperature monitoring unit 34 at high speed among the baths 50and 51 to expose them alternately to the different temperatures in thebaths 50 and 51 as required for the thermal cycling. The reactortransfer mechanism 85 is comprised of an X stage 86 moving along an Xaxis linear guide 87 for the reactor 15 and the temperature monitoringunit 34 to reach to a region above the baths 50 and 51, and a Z stage 88moving along a Z axis linear guide 89 for the reactors 15 to move themdown to enter the bath medium 75 or to be withdrawn from the bath medium75, as shown by the dashed path 200. The reactor 15 has an opening forloading and the reaction material 21 and the opening is sealable.

Increasing the speed of thermal cycling has been a constant challengefor the industry. The present invention provides an improved apparatusfor enabling thermal cycling nucleic acid at an increased speed ataffordable cost without using complex and expensive components orconsumables. The apparatus is robust, light weight, easy to use, needs asmall amount of bath medium in the baths and can handle disposablereactors for the reaction material to avoid cross contamination from onereactor to the next. This invention provides a great positive impact onbiological analysis.

SUMMARY

Unless specified otherwise, the term “comprising” and “comprise” andgrammatical variants thereof, are intended to represent “open” or“inclusive” language such that they include recited elements but alsopermit inclusion of additional, unrecited elements. The terminologies‘first bath’, ‘second bath’ . . . ‘sixth bath’ do not constitute thecorresponding number of baths in a sequence but merely are names forease of identification with respect to the purpose they serve. Thesebaths may not necessarily represent separate physical entities as someof them may be shareable. The term ‘thermal processing’ includes: a)thermal cycling, and optionally includes: b) thermal process stepsbefore and/or after thermal cycling. The term ‘thermal profile’ refersto the temperature-time variation of the reactor(s) during a) alone orduring a) with b).

According to a first aspect, apparatus for thermal processing of nucleicacid in a thermal profile is provided. The apparatus employs a reactorholder for holding reactor(s) to accommodate reaction materialcontaining nucleic acid and the reactor(s) being in any form such astube(s) or wellplate(s) or chip(s) or cartridge(s), at least two bathsseparated by thermally insulating partition plate(s) where thereactor(s) is/are allowed to attain a predetermined temperature; and atransfer means for allowing the reactor(s) to change position once orplurality of times between any two adjacent baths of the at least twobaths by selectively opening the partition plate(s) to open in ahorizontal direction or a vertical direction and without lifting thereactor(s) out of the baths. Since this feature of employing thepartition plate allows transfer of the reactor(s) between the bathswithout going through the step of lifting up the reactors from thebaths, the thermal processing is possible at a faster rate. Herein, thereactors can be lowered into any bath before initiating the thermalprocessing and thereafter lifted up from any bath after the thermalprocessing. The thermally insulating nature of the partition plate helpsto maintain the bath temperatures on either side. The partition plateremains in the open position only for a minimal duration for thereactor(s) to change baths.

According to a preferred embodiment, the at least two baths comprise afirst bath where the reactor(s) is/are allowed to attain a predeterminedhigh target temperature T_(HT), wherein the T_(HT) is in the region85-99 degree Celsius for pre-denaturation and denaturation of thenucleic acid; and a second bath where the reactor(s) is/are allowed toattain a predetermined low target temperature T_(LT), wherein the T_(LT)is in the region 45-75 degree Celsius for annealing of primers or probesonto the nucleic acid or for primer extension for thermal cycling thereactor(s) to attain polymerase chain reaction (PCR) amplification orprimer extension. These are typical temperature ranges for PCR thermalcycling.

According to an embodiment, a third bath the reactor(s) is/are allowedto attain a predetermined medium target temperature T_(MT), wherein theT_(MT) is for annealing of primers or probes onto nucleic acid.According to another embodiment a fourth bath, the reactor(s) is/areallowed to attain a predetermined medium target temperature T_(MT),wherein the T_(MT) is for extension of primers on nucleic acid. Thisfeature further enhances the speed of thermal cycling engaging more thantwo baths. Herein, the reactors may be lowered into any of the baths asdesired before initiating the thermal cycle and thereafter lifted-offfrom any of the baths after the thermal cycling. The T_(MT) may be usersettable to T_(LT) for achieving a desired thermal profile.

The apparatus may further comprise a fifth bath where the reactor(s)is/are allowed to attain a temperature T_(AP) for an additional processfor the reactor(s) before thermal cycling, the additional process beingone from the group consisting reverse transcription-polymerase chainreaction (RT-PCR), hot start process and isothermal amplificationreaction. The apparatus may further comprise a sixth bath that can beprogressively heated while conducting melt curve analysis after thermalcycling. The fifth and the sixth baths allow the thermal cycling to beintegrated with the previous and the following steps respectively whileadvantageously using the inventive concept to reduce the time forthermal processing. Integrating the whole process in a single apparatushelps in automating the processing line and also overcomes therequirement of providing controlled ambience and trained personnel forconducting these extra steps. The reactor(s) may be allowed to stabilizein any of the baths if desired for the thermal profile.

The bath medium in any of the baths may be in any phase including air,liquid, solid, powder and a mixture of any of these. The reactor(s)is/are preferably oriented to allow minimum surface area to be in thedirection of movement between the baths, in order to lower theresistance from the bath medium so that faster movement is possiblethereby increasing the speed of thermal cycling and reducing powerconsumption. The minimum surface also advantageously minimizesdisturbance to the bath mediums when the reactor(s) move between thebaths with the partition plate(s) in the open position, thus creatingminimal disturbance in the temperatures of the baths.

The present invention enables the entire process of thermal processingincluding thermal cycling to be completed in a significantly shortertime due to the usage of the partition plates.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, same reference numbers generally refer to thesame parts throughout. The drawings are not to scale, instead theemphasis is on describing the concept.

FIG. 1A is a cross-sectional elevation view of a typical set up in theart for thermal cycling a reaction material containing nucleic acid;

FIG. 1B is a cross-sectional elevation view of a set up for thermalcycling a reaction material containing nucleic acid according to anembodiment of the invention;

FIG. 2A to FIG. 2F are plan views of the movable partition plateseparating the high temperature and the low temperature baths in a 2step PCR process, according to an embodiment of the invention;

FIG. 2A shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the reactors arein the high temperature bath and the partition plate is at a closeposition, separating the hot and cold baths;

FIG. 2B shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the partitionplate moves to an open position after the reactors have been in bath fora predetermined time;

FIG. 2C shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the reactorsspeedily move to the low temperature bath.

FIG. 2D shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the partitionplate moves to the close position after the reactors moved to the lowtemperature bath;

FIG. 2E shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the partitionplate moves to the open position again after the reactors have been inlow temperature bath for another predetermined time;

FIG. 2F shows the plan view of the movable partition plate separatingthe high temperature and the low temperature baths when the reactorsmove back to the hot bath and the partition plate closes thereafter (notshown) to complete one thermal cycle of a 2-step PCR thermal cyclingprocess;

FIGS. 3A to 3D are plan views of the movable partition plates separatingthe high, the medium and the low temperature baths for a 3 step PCRprocess, according to an embodiment of the invention;

FIG. 3A shows plan view of the movable partition plates separating thehigh, the medium and the low temperature baths for a 3 step PCR processwhen the reactors are in the high temperature bath;

FIG. 3B shows plan view of the movable partition plates separating thehigh, the medium and the low temperature baths for a 3 step PCR processwhen the reactors are in the medium temperature bath;

FIG. 3C shows plan view of the movable partition plates separating thehigh, the medium and the low temperature baths for a 3 step PCR processwhen the reactors are in the low temperature bath;

FIG. 3D shows plan view of the movable partition plates separating thehigh, the medium and the low temperature baths for a 3 step PCR processwhen the reactors are back to the high temperature bath;

FIG. 4 is a plan view of the movable partition plates separating fivebaths, according to an embodiment of the invention:

FIG. 5A illustrates an exemplary thermal profile wherein the profile forthermal cycling is achievable by the configuration at FIG. 2:

FIG. 5B illustrates an exemplary thermal profile wherein the profile forthermal cycling is achievable by the configuration at FIG. 3;

FIG. 6A is a perspective view of an exemplary reactor in the form ofchips that is also usable with the apparatus under the presentinvention;

FIG. 6B is a perspective view of another exemplary reactor in the formof chips that is also usable with the apparatus under the presentinvention; and

FIG. 7 is a cross-sectional elevation view of a reactor with opticalfibres for illuminating the reaction material and collecting the emittedlight for analyses.

DETAILED DESCRIPTION

The following description presents several preferred embodiments of thepresent invention in sufficient detail such that those skilled in theart can make and use the invention.

FIG. 1B shows a schematic view of an embodiment of a portion of thethermal cycling apparatus for thermal cycling of nucleic acid such asfor PCR, primer extension or other enzymatic reactions. The apparatushas two baths 50 and 51 each containing the bath medium 75, a bathheater 17 and a bath temperature sensor 39 mounted along the bathsurface to enable control of the temperature of the bath medium 75. Thebath temperature sensors 39 may be positioned inside the baths 50, 51 asshown. In this embodiment, the bath 50 is suitable for the step ofdenaturation and the bath 51 is suitable for the step of annealingand/or extension. The cooler 16 is useful when the bath 51 needs to beactively cooled to below room temperature. Active cooling device such asa thermoelectrical cooler or a fan can also be installed. The bathmedium 75 shown here is liquid, however any other type of fluid orpowder or solid bath medium 75 may also be used. For some embodiments,the bath heater 17 on the low temperature bath 51 is optional, if bath51 does not have to be heated over room temperature. For the thermalcycling, the reactor 15 is alternately transferred between the baths 50,51 multiple times. To enable fast movement of the reactor 15 in the bathmedium 75, slim reactor 15 is preferable such as glass capillaries. Thereactor 15 is sealed with a sealant or a cap 77 and a portion of thereactor 15 herein is transparent (not shown) to allow light to passthrough for dye or probe excitation and fluorescence imaging. Atemperature monitoring unit 34 is installed on the reactor holder 33that moves along with the reactor 15 between the baths 50, 51. Thetemperature monitoring unit 34 contains a fast response temperaturesensor 38 inside. The temperature monitoring unit 34 has a shape similarto that of the reactor 15 and is constructed to have a similar or thesame steady state and transient thermal characteristics as those of thereactor 15, so that the temperature reading and thermal response issimilar or same as those of the reactor 15 unless another reactor 15itself is used for the purpose. For example, the temperature monitoringunit 34 may have the fast response temperature sensor 38 inserted intowater or oil or a layer of oil over water 22 and sealed. The reactortransfer mechanism 85 transfers the reactor 15 and the temperaturemonitoring unit 34 at high speed among the baths 50 and 51 to exposethem alternately to the different temperatures in the baths 50 and 51 asrequired for the thermal cycling. The reactor transfer mechanism 85comprises an X stage 86 moving along an X axis linear guide 87 for thereactor 15 and the temperature monitoring unit 34 to reach to a regionabove the baths 50 and 51, and a Z stage 88 moving along a Z axis linearguide 89 for the reactor 15 to move them down to enter the bath medium75 or to be withdrawn from the bath medium 75. The reactor 15 has anopening for loading the reaction material 21 and the openings aresealable. The sealant 77 may be made of a silicone rubber or UV curedpolymer, hot melt and/or wax and/or gel which is in solid phase duringthermal cycling. The sealing can also be achieved using liquid such asoil, viscous polymer, and gel. The highly viscous liquid can be appliedto the opening and/or top section of the reactor 15 to block the vaporgenerated from the reaction material 21 from leaking out. Thearrangement for the fluorescent imaging may be in any form as in theart. Herein, the bath 51 has a transparent window 25 so thatillumination from the illumination source 44 reaches the reactor 15inside and the emitted beam is received by the receiver 43. Thepartition plate 316 within the two baths 50, 51 opens for a minimalduration that is just adequate for the reactor transfer mechanism 85 tooperate the X stage 86 for enabling the reactors 15 to change positionbetween the baths 50, 51, without operating the Z stage 88 asillustrated by the double headed dashed arrow 200. On both sides of thepartition plate 316, the levels of the bath mediums 75 are maintainedsubstantially the same, hence the flow of the bath mediums 75 betweenthe baths 50, 51 is negligible and the heat diffusion between the twobaths 50, 51 is also minimized. The partition plate 316 being thermallyinsulating, the temperatures of the baths 50, 51 are better maintained.When the bath medium 75 is a high thermal conductivity metallic powder,the flow of the bath mediums 75 between the baths 50, 51 is even lesseras compared to the case of liquid.

FIGS. 2A-2F show plan views of the high temperature bath 50 and the lowtemperature bath 51 for a 2-step thermal cycling process. The baths 50,51 are separated by a partition plate 316 that is made of a thermallyinsulating material. At (a), the reactors 15 are in the high temperaturebath 50 and the partition plate 316 is at close a position, separatingthe hot and cold baths 50, 51. At (b), the partition plate 316 moves toan open position after the reactors 15 have been in bath 50 for apredetermined time. At (c), the reactors 15 speedily move to the lowtemperature bath 51 and promptly the partition plate 316 moves to theclose position as shown at (d). At (e) the partition plate 316 moves tothe open position again after the reactors 15 have been in bath 51 foranother predetermined time. At (f) the reactors 15 move back to the hotbath 50 and the partition plate 316 closes thereafter (not shown) tocomplete one thermal cycle of a 2-step PCR thermal cycling process. Theline arrows illustrate the movement of the partition plate 316 and theblock arrows illustrate the movement of the reactors 15. In thisembodiment, in the bath 50 the reactors 15 are allowed to attain apredetermined high target temperature T_(HT), wherein the T_(HT) is inthe region 85-99 degree Celsius for pre-denaturation and denaturation ofthe nucleic acid. In the bath 51, the reactors 15 are allowed to attaina predetermined low target temperature T_(LT), wherein the T_(LT) is inthe region 45-75 degree Celsius for annealing the nucleic acid. Theseare typical temperature ranges for PCR thermal cycling.

FIGS. 3A to 3D show plan views of the high 50, the low 51 and the medium52 temperature baths for a 3-step thermal cycling process, with twopartition plates 316, 317, the mechanism of operation of the partitionplates being similar to the previous embodiment at FIGS. 2A-2F. Thepartition plates 316 and 317 open and close at the fastest possiblespeed that the electro-mechanics of the apparatus allows so that thebath mediums 75 from the either side do not substantially diffuse intothe opposite sides. Any small diffusion alters the temperatures of theadjacent baths slightly which are compensated by the heaters 17 and thecoolers 16 as applicable. The figures show the reactors 15 in the bath50, followed by bath 52, followed by bath 51 and then back to bath 50 inone thermal cycle. The transfer mechanism 85 may be appropriatelyutilized for allowing the reactors 15 to change position between any twoadjacent baths for thermal cycling without lifting the reactors 15 outof the baths when the partition plate 316, 317 between the adjacentbaths is in an open position. In (a) to (d) respectively the reactors 15are in the high temperature bath 50, in the medium temperature bath 52,in the low temperature bath 51 and then back to the high temperaturebath 50 when both the partition plates open for short durations for thereactor(s) 15 to move to the high temperature bath 50 by speedilycrossing through the medium temperature bath 52. In this embodiment, inthe bath 52 the reactors 15 are allowed to attain a predetermined mediumtarget temperature T_(MT), wherein the T_(MT) is for annealing ofprimers or probes onto nucleic acid. Alternately in the bath 52 thereactors 15 are allowed to attain a predetermined medium targettemperature T_(MT), wherein the T_(MT) is for extension of primers onnucleic acid.

FIG. 4 shows a plan view of the high 50, the low 51 and the medium 52temperature baths for a 3-step thermal cycling process, with twopartition plates 316, 317, the mechanism of operation of the partitionplates being similar to the previous embodiment at FIGS. 3A-3D. In thisembodiment, an additional process bath 53 and a melt curve analysis bath54 are also shown along with the respective partition plates 318 and319. The additional process bath 53 allow the reactors 15 to attain atemperature TA before the thermal cycling, the additional process beingone from the group consisting reverse transcription-polymerase chainreaction (RT-PCR), hot start process and isothermal amplificationreaction. The melt curve analysis bath 54 can be progressively heatedwhile conducting fluorescent imaging for the reaction material 21 in thereactors 15 while in the bath 54. As shown by the block arrow, thereactors 15 after being in the bath 53 for a predetermined time is movedto bath 50 in a single step by opening the partition plate 318 for ashort duration. Thereafter, the reactors 15 are cycled between the baths50, 52, 51 several times as shown by the multiple arrows and asdescribed under FIGS. 3A-3D. Once the thermal cycling is completed, thereactors 15 are moved to the bath 54 as shown by the dashed arrow byopening the partition plate 319 for a short duration, for the melt curveanalysis while progressively heating the bath. All these movements forthe reactors 15 to change the baths are conducted without lifting-up thereactors 15 from the baths. The process of thermal cycling may thus bepreceded by any additional thermal process and may be followed by meltcurve analysis in an integrated apparatus while advantageously using theinventive concept of employing the partition plates 316 to 319, toreduce the time of thermal processing. The invention may beadvantageously used with any number of baths with the required number ofpartition plates by positioning the baths and the partition plates in anappropriate configuration so that the transfer means 85 allows thereactors 15 to change the baths only by the X-stage 86.

FIG. 5A is an exemplary time-temperature graphical representations oftypical 2-step thermal cycling process followed by a melt curveanalysis. Only three cycles are shown over the processes of denaturationand annealing employing two baths 50, 51. After the thermal cycling, thereactor 15 are placed in bath 54 with at least a partially transparentbath medium 75 that is progressively heated while melt curve analysis isconducted. The fluorescence signals from the reactor 15 are acquired atmultiple temperatures to form a fluorescence-temperature curve for meltcurve analysis. The bath medium 75 for the melt curve analysis may beair or transparent liquid and the bath needs to have a transparentwindow 25. Both the bath medium 75 and the window 25 are required tohave low auto-fluorescence. In this embodiment, the temperature of thebath medium 75 in the high temperature bath 50 is maintained at atemperature T_(HT) and the temperature of the bath medium 75 in the lowtemperature bath 51 is maintained at a temperature T_(LT). This kind ofthermal profile for thermal cycling is achievable by the bathconfiguration shown at FIGS. 2A-2F. The bath for the melt curve analysisis not shown where the reactor(s) 15 may be placed by the transfermechanism 85 and by using another partition plate. FIG. 5B is anexemplary time-temperature graphical representations of typical 3-stepthermal cycling process followed by a melt curve analysis. This kind ofthermal profile for thermal cycling is achievable by the bathconfiguration shown at FIG. 3A-3D or 4. Herein the profile in the bath53 has not been shown which may typically be in the range of 40-75degree Celsius. Only three cycles are shown over the processes ofdenaturation, annealing and extension employing the three baths 50, 51,52. After the thermal cycling, the melt curve analysis is conducted asdescribed under FIG. 5A. The reactors 15 are placed in the mediumtemperature bath 52 for a longer duration to stabilize at T_(MT).Likewise, any other kind of thermal profile can be achieved byappropriately operating the partition plates 316, 317 and the X stage86. Stabilization at T_(HT) and T_(LT) may also be obtained (not shown)by placing the reactors 15 in the baths 50 and 51 respectively forlonger durations.

FIG. 6A is a biocchip 31 consisting of reactors 15 in the form of wells.The reaction material 21 is dispensed from the opening of the reactors15 and sealed by a cover or sealing fluid 30. The biochip 31 is thenmounted onto the reactor holder 33. FIG. 6B is a perspective view of thereactors 15 being accommodated in a biochip 31 for use in the baths.Herein, the reactors 15 are arranged in the biochip 31. There is atleast one inlet 313 which is in fluid communication with the reactors 15via a network of channels 315. The reaction material 21 to be tested canbe loaded into the inlet 315 that subsequently flow into the reactors15.

FIG. 7 shows an embodiment where the reactor 15 is made of a metaltubing. It is provided with an optical fiber 309 for light transmissionfrom an illumination light source such as an LED (not shown) into thereaction material 21 inside the reactor 15. Optical fiber 310 is forlight transmission from the reaction material 21 to a photodetector (notshown). This facilitates the optical detection for the reactor body evenwhen non-transparent. Besides, it also helps to conduct the imagingwhile the reactors 15 are in any of the baths with transparent ornon-transparent medium. The reactors 15 with this arrangement need notbe made stationary for imaging thereby increasing the speed of theprocess of the thermal cycling.

The bath medium 75 may comprise one or more selected from a groupconsisting of water, oil, glycerin, chemical liquid, liquid metal, gas,air, metal powder and silicon carbide powder and/or beads and theirmixture. The materials used to construct the reactors 15 may beplastics, elastomer, glass, metal, ceramic and their combinations, inwhich the plastics include polypropylene and polycarbonate. The glassreactor 15 can be made in a form of a glass capillary of small diameterssuch as 0.1 mm-3 mm OD and 0.02 mm-2 mm ID, and the metal can bealuminum in form of thin film, thin cavity, and capillary. Reactormaterials can be made from non-biological active substances withchemical or biological stability. At least a portion of the reactor 15is preferred to be transparent. The volume of the at least one reactor15 may be in the range 1 μL to 500 μL. Smaller the volume, faster is theheat transfer, higher is the speed of PCR, smaller are the required bathsizes and more compact is the apparatus. The reaction material in allthe reactors 15 in the reactor holder 33 may not be identical.Simultaneous PCR can be advantageously conducted for different materialsif the bath temperatures are suitable. The baths may be shared betweendifferent process steps by altering the temperatures. The embodimentsdescribed above may be suitable for one reactor 15 or a pluralityreactors 15. The reactor 15 may be in the form of tube(s) as shown or aswellplate(s) or chip(s) or cartridge(s) and the like.

According to an embodiment, the apparatus facilitates DNA melt curveanalysis at the end of a PCR thermal cycle. Herein, after the DNAamplification process using PCR thermal cycling or other techniques, thereactors 15 are transferred to a bath (not shown) containing atransparent liquid bath medium 75. Such a bath has at least a portion toallow light to pass for illumination of the reactors 15 inside the bathand fluorescent imaging of the reactors 15. The bath medium 75 iscapable of being heated up progressively while imaging the reactors 15from a low temperature to a high temperature covering all the melttemperatures of the amplicons in the reaction materials 21 in thereactors 15. In the entire heating process, fluorescence signals fromthe reactors 15 are acquired at multiple temperatures to form afluorescence-temperature curve for the melt curve analysis.

When using the above described apparatus for nucleic acid analysis andprocessing, the reaction material 21 comprises reaction constituentsincluding at least one enzyme, nucleic acid and/or particle containingat least one nucleic acid, primers for PCR, primers for isothermalamplifications, primers for other nucleic acid amplifications andprocessing, dNTP, Mg²⁺, fluorescent dyes and probes, control DNA,control RNA, control cells, control micro-organisms, and other reagentsrequired for nucleic acid amplification, processing, and analysis. Theparticle containing nucleic acid mentioned above comprises at least onecell virus, white blood cell and stromal cell, circulating tumor cell,embryo cell. One application may be to use the apparatus to testdifferent kinds of reaction materials 21 against the same set of primerand probes, such as test more than one sample. For such application,different kinds of reaction material 21 containing no target primersand/or probes are each loaded into one reactor 15 in a reactor array,with all the reactors 15 being pre-loaded with the same set or the samesets of PCR primers and/or probes. For the same application, differentkinds of reaction materials 21 pre-mixed with respective PCR targetprimers and/or probes are each loaded into one reactor 15 in a reactorarray, with all the reactors 15 being not pre-loaded with the same setof PCR primers and or probes. The reaction materials 21 can includecontrol genes and/or cells and corresponding fluorescent dyes or probes.In the above situations, the different probes emit light of differentwavelengths. Another application of the methods and devices are used totest the same reaction material 21 against different sets of primer andprobes. One example of such an application is to test one type of samplefor more than one purpose. For this application, a single reactionmaterial 21 is added into the reactors 15 each loaded with at least onedifferent set PCR primers and or probes. The reaction material 21 caninclude control genes and/or cells and corresponding fluorescent dyes orprobes. In the above situations, the different probes emit light ofdifferent wavelengths. The above reaction material 21 is used inpolymerase chain reaction, reverse transcription-PCR, end-point PCR,ligase chain reaction, pre-amplification or target enrichment of nucleicacid sequencing or variations of polymerase chain reaction (PCR),isothermal amplification, linear amplification, library preparations forsequencing, bridge amplification used in sequencing. The variation ofthe polymerase chain reaction mentioned above comprises reversetranscription-PCR, real-time fluorescent quantitative polymerase chainamplification reaction and real-time fluorescent quantitative reversetranscription polymerase chain amplification reaction, inversepolymerase chain amplification reaction, anchored polymerase chainamplification reaction, asymmetric polymerase chain amplificationreaction, multiplex PCR, colour complementation polymerase chainamplification reaction, immune polymerase chain amplification reaction,nested polymerase chain amplification reaction, the target enrichment ofpre-amplification or nucleic acid sequencing, ELISA-PCR.

When the apparatus is in operation, the partition plate may open in ahorizontal direction or in a vertical direction. Any other directions isalso possible. In embodiments where the partition plate opens and closesin the horizontal direction and needs to protrude out of the bath areathrough a slot or a narrow cavity, in operation the slot or the cavitytends to get partially clogged with powder when the powder is used asthe bath medium. Such a scenario offers increased resistance to themovement of the partition plate through the slot or the cavity. Thismakes the movement of the partition plate difficult.

According to an alternate embodiment, the partition plate opens andcloses in the vertical direction, by lifting the partition plate up inthe air and placing it down back to the original position, withouthaving to move it through any such powder filled slot or cavity. Thereis however no such issue of partially clogging expected with the slot orthe cavity when the bath medium is a liquid instead of a powder.

Any other mechanism for opening and closing the partition plate isequally possible.

The reactors may be in any form, such as tubes or wellplates or chips orcartridges. The tubes include capillaries.

From the foregoing description it will be understood by those skilled inthe art that many variations or modifications in details of design,construction and operation may be made without departing from thepresent invention as defined in the claims.

What is claimed is:
 1. An apparatus for thermal processing of nucleicacid in a thermal profile, the apparatus employing a reactor holder forholding reactor(s) to accommodate reaction material containing nucleicacid, the apparatus comprising: at least two baths separated bythermally insulating partition plate(s) where bath mediums in use areeach maintainable at a predetermined temperature; and a transfer meansfor allowing the reactor(s) to change position once or plurality oftimes between any two adjacent baths of the at least two baths byselectively opening the partition plate(s) and without lifting thereactor(s) out of the baths.
 2. The apparatus according to claim 1,wherein the at least two baths comprise: a first bath where thereactor(s) is/are allowed to attain a predetermined high targettemperature T_(HT), wherein the T_(HT) is in the region 85-99 degreeCelsius for pre-denaturation and denaturation of the nucleic acid; and asecond bath where the reactor(s) is/are allowed to attain apredetermined low target temperature T_(LT), wherein the T_(LT) is inthe region 45-75 degree Celsius for annealing of primers or probes ontonucleic acid or for primer extension, the first and the second bathsbeing for thermal cycling the reactor(s) to attain polymerase chainreaction (PCR) amplification or primer extension.
 3. The apparatusaccording to claim 2, further comprising: a third bath where thereactor(s) is/are allowed to attain a predetermined medium targettemperature T_(MT), wherein the T_(MT) is for annealing of primers orprobes onto nucleic acid.
 4. The apparatus according to claim 2, furthercomprising: a fourth bath where the reactor(s) is/are allowed to attaina predetermined medium target temperature T_(MT), wherein the T_(MT) isfor extension of primers on nucleic acid.
 5. The apparatus according toclaim 2, further comprising: a fifth bath where the reactor(s) is/areallowed to attain a temperature T_(AP) for an additional process for thereactor(s) before thermal cycling, the additional process being oneselected from the group consisting of a) reversetranscription-polymerase chain reaction (RT-PCR), b) hot start process,and c) isothermal amplification reaction.
 6. The apparatus according toclaim 2, further comprising: a sixth bath that can be progressivelyheated while conducting melt curve analysis after thermal cycling. 7.The apparatus according to claim 6, wherein the reactor(s) is/areallowed to stabilize in the first bath, the second bath, the third bath,the fourth bath, the fifth bath or the sixth bath.
 8. A method of usingthe apparatus of claim 3, the method comprising: setting the T_(LT)equal to the T_(MT).
 9. A method of using the apparatus of claim 1, themethod comprising: using the bath medium in at least one phase selectedfrom the group consisting of air, liquid, solid, and powder.
 10. Themethod according to claim 9, further comprising: orienting thereactor(s) held by the reactor holder to allow minimum surface area ofthe reactor(s) to be in a direction of movement of the reactor(s)between the baths.
 11. A method of using the apparatus of claim 4, themethod comprising: setting the T_(LT) equal to the T_(MT).
 12. Theapparatus according to claim 1, wherein in operation, the partitionplates open along a horizontal direction.
 13. The apparatus according toclaim 1, wherein in operation the partition plates open along a verticaldirection.